LARGE-SCALE IMMUNOAFFINITY PURIFICATION OF RECOMBINANT SOLUBLE HUMAN-ANTIGEN CD4 FROM ESCHERICHIA-COLI-CELLS

A large-scale immunoaffinity (IA) purification process was developed f or the isolation of recombinant soluble antigen CD4 (sCD4) from Escher ichia coil fermentations. The monoclonal antibody used for LA purifica tion of sCD4 recognized a conformation-dependent epitope on the surfac e of domain 1 of CD4. IA chromatography was used to purify both sCD4-1 83, consisting of the N-terminal 183 amino acids of human CD4, and sCD 4-PE40, a fusion protein consisting of the N-terminal 178 amino acids of CD4 and amino acids 1-3 and 253-613 of Pseudomonas exotoxin A (PE40 ). sCD4-183 was purified from E. coil cell pellets using cell disrupti on, protein solubilization, oxidation, Q-Sepharose anion-exchange and LA chromatography steps. sCD4-PE40 was purified from cell pellets usin g cell disruption, protein solubilization, oxidation, Cu2+-immobilized metal-affinity chromatography, anion-exchange and IA chromatography s teps. The IA-purified sCD4 analogues demonstrated the correct apparent molecular masses on SDS/PAGE. The immobilized monoclonal antibody app eared to select for correctly folded CD4 protein, since sCD4-183 and s CD4-PE40 purified by the IA method bound human-immunodeficiency-virus glycoprotein gp120 (HLV gp120) in vitro. sCD4-PE40 purified by IA chro matography also inhibited protein synthesis in CV-1 cells expressing H IV gp120/160 at the cell surface. Relatively high recoveries of sCD4-1 83 and sCD4-PE40 were observed in the LA step of the purification proc ess (71 and 79% recovery respectively). The results demonstrate that i mmobilized monoclonal antibodies directed against conformational epito pes may be used for rapid purification of gram amounts of correctly fo lded protein from mixtures of oxidized E, coil proteins.